P1384: MULTI-EFFECTOR IMMUNE CELL RETARGETING USING A BISPECIFIC SINGLE-CHAIN VARIABLE FRAGMENT LIBRARY IN AGGRESSIVE B CELL LYMPHOMAS
Authors: Max Freihammer, Pramudini Jayalath, Zhiyuan Shi, Jennifer Schitz, Verena Kern, Meike Schwandt, Till Braun, Olivia Käsgen, Jan-Michel Heger, Sven Borchmann
Journal: HemaSphere (2023)

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: Various bispecific antibodies (bsAbs) in different designs have been developed to treat patients with aggressive B cell lymphomas such as diffuse large B cell lymphoma (DLBCL or classic Hodgkin lymphoma (cHL) (Budde et al. J Clin Oncol, 2022). Despite effective first-line treatments, the prognosis of these patients remains relatively poor in the relapsed/refractory (r/r) situation. BsAbs that recruit immune effector cells to tumor cells have improved the treatment of DLBCL in particular (Hutchings et al. Lancet, 2021) and might be an alternative to other immunotherapeutic approaches such as CAR-T cells that can be associated with a long and cumbersome manufacturing process and high cost. With few exceptions, current BsAbs target only one tumor-associated target antigen and one immune effector cell antigen. Aims: Here, we aim to develop a library of bispecific single-chain variable fragments (ScFvs) targeting CD19 and CD30 positive hematological malignancies by combined engagement of multiple effector cell antigens on T cells and NK cells. Methods: We produced a bispecific scFv library targeting CD19 and CD30 on lymphoma cells and CD3, CD16 and CD28 on immune effector cells. A schematic of the design is shown in Fig. 1A. Protein production is performed using an optimized mammalian protein expression system. The bispecific scFvs are subsequently recovered through an optimized, tag-based purification process. Flow cytometry is used to show specific binding to tumor and immune effector cells. In vitro cytotoxicity (TOX) assays have been conducted to investigate the potential of the bispecific scFvs in our library to induce tumor cell killing in a dose-dependent manner. Further functional assays, such as cytokine release studies of effector cells after bispecific scFvs-mediated target cell engagement, have been performed as well. Results: The binding of the CD30-targeting bispecific scFvs from our library was shown to be specific for the CD30-expressing L540 cell line. Likewise, the CD19-targeting bispecific scFvs from our library were shown to specifically bind the CD19-expressing Raji cell line. Exemplary flow cytometry experiments are shown in Fig. 1B. Further flow cytometry studies showed binding to the respective immune effector cell antigens (CD3 and CD28 on T cells, CD16 on NK cells). Furthermore, we were able to show dose-dependent and target-specific cytotoxicity for all our designs, with cytotoxicity of CD3- and CD16-targeting bispecific scFvs consistently higher than CD28-targeting bispecific scFvs. Cytotoxicity was specific for the respective CD19- or CD30-expressing target cells. Additionally, we observed synergy between CD3-, CD16- and CD28- targeting bispecific scFv when tested together in cytotoxic assays, suggesting that simultaneous multi-effector immune cell retargeting with multiple scFvs might be beneficial. Exemplary cytotoxicity data from the selected experiments is shown in Fig. 1C. Further functional validation data will be presented at the meeting. Summary/Conclusion: We have established a platform for the production of a library of bispecific scFvs and are able to show dose-dependent and target-specific binding, cytotoxicity and expected in vitro functionality. We were able to show that we can combine multiple scFvs targeting immune effector cell antigens on different effector cells, such as T and NK cells, and achieve synergy in in vitro experiments. Next, in vivo studies are planned for further preclinical validation of our approach.Keywords: Bispecific, B cell lymphoma, CD30, Cancer immunotherapy